Enhancing Tumor Cell Response to Multidrug Resistance with pH-Sensitive Quercetin and Doxorubicin Conjugated Multifunctional Nanoparticles

Accepted Manuscript Title: Enhancing Tumor Cell Response to Multidrug Resistance with pH-Sensitive Quercetin and Doxorubicin Conjugated Multifunctional Nanoparticles Author: Cenk Daglioglu PII: DOI: Reference:

S0927-7765(17)30270-9 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.05.012 COLSUB 8541

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

20-2-2017 28-4-2017 6-5-2017

Please cite this article as: C. Daglioglu, Enhancing Tumor Cell Response to Multidrug Resistance with pH-Sensitive Quercetin and Doxorubicin Conjugated Multifunctional Nanoparticles, Colloids and Surfaces B: Biointerfaces (2017), http://dx.doi.org/10.1016/j.colsurfb.2017.05.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title:

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Enhancing Tumor Cell Response to Multidrug Resistance with pH-Sensitive Quercetin and Doxorubicin Conjugated

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Multifunctional Nanoparticles

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Author:

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Cenk Daglioglu

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Address:

Izmir Institute of Technology, Faculty of Science, Department of Molecular Biology and

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Genetics, Urla/Izmir 35430, Turkey

Office Phone: +90 232 750 7319 Fax: +90 232 750 7303

e-mail: [email protected]

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Abstract Classical chemotherapy uses chemotherapeutic agents as a mainstay of anticancer treatment. However, the development of multidrug resistance to chemotherapy limits the effectiveness of

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current cancer treatment. Nanosized bioconjugates combining a chemotherapeutic agent with a pharmacological approach may improve the curative effect of chemotherapeutic agents.

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Herein I addressed this issue by describing the synthesis, and testing of, pH-responsive

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Fe3O4@SiO2(FITC)-BTN/QUR/DOX multifunctional nanoparticles. The particles were designed to modulate resistance-mediating factors and to potentiate the efficacy of DOX

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against chemoresistance. The physicochemical properties of the nanoparticles were characterized based on the combination of several techniques: dynamic light scattering (DLS),

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zeta-potential measurement, Fourier transform infrared spectroscopy (FTIR), electron microscopy techniques (SEM and STEM with EDX) and an in-vitro pH-dependent release

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study. Cellular uptake and cytotoxicity experiments demonstrated enhanced intracellular

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delivery and retention of nanoparticles in the cytoplasm and efficient reduction of cancer cell

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viability in drug-resistant lung carcinoma A549/DOX cell lines. This did not affect internalization and viability of an immortalized human lung epithelial cell line BEAS-2B. Moreover, proapoptotic and antiproliferative studies showed that Fe3O4@SiO2(FITC)BTN/QUR/DOX nanoparticles can promote apoptosis, inhibit tumor cell proliferation, and enhance the chemotherapeutic effects of DOX against multidrug resistance. These results confirm that this multifunctional platform possesses significant synergy between QUR and DOX and is promising for development as an antitumor treatment in cancer therapy.

Keywords:

Multifunctional

nanoparticles,

Bioconjugation,

Multidrug

resistance,

Combination cancer therapy, Drug delivery

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Introduction Multidrug resistance (MDR) to chemotherapeutic agents is one of the major unsolved problems for the success of cancer chemotherapy.1 A number of different mechanisms, either

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inherent or acquired, can mediate the development of MDR against every effective anticancer drug. These mechanisms include decreased drug uptake, increased drug efflux, activation of 2

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detoxifying systems, superior DNA repair mechanisms and defective apoptotic pathways.

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The etiology of MDR may be multifactorial, but the classic resistance that develops in cancer cells has most often been linked to the overexpression of P-glycoprotein (P-gp), an ATP-

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dependent membrane transporter that acts as a drug efflux pump.3 Administration of chemotherapy drugs triggers increased expression of these transporters, thus effectively

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lowering intracellular concentrations of the chemotherapy drugs in tumor cells. To produce adequate intracellular concentrations of the chemotherapeutic, higher serum dosage is

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required but results in greater toxicity on normal tissues.4 Therefore, a promising strategy to

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enhance the efficacy of classical chemotherapy is antagonization of resistance-mediating

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factors in order to restore drug sensitivity.

Modulating resistance-mediating factors as a way of reversing MDR has been extensively studied in recent years, which led to the discovery of many P-gp inhibitors for the treatment of cancers.5 Investigations to eliminate the major limitation of the early agents in decreasing their unacceptable toxicity and unfavorable pharmacokinetic interactions prompted the development of less toxic and more potent inhibitors.6 Although the adverse effects of these inhibitors disappeared after the introduction of third-generation agents, recent clinical trials showed disappointing results due to their toxicity and lack of pharmacological effects.7 Therefore, investigation of natural products with potential MDR modulatory activity is highly anticipated. In this context, the natural flavonoid quercetin (QUR) drew much attention because epidemiological studies suggest that dietary intake of certain foods rich in flavonoids, 3 Page 3 of 36

including quercetin, have preventive activity against a broad range of cancers.8 To date, quercetin has been reported to possess a broad spectrum MDR modulation activity. These activities are diverse and include: mimicking the adenine moiety of ATP-binding site of the

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ABC transporters to inhibit efflux of the drugs; accelerating TNF-alpha induced growth inhibition; inducing death receptor 5 (DR5)-mediated apoptosis; and suppressing of Akt-

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mediated antiapoptotic survivin expression.9-12 Despite the multifaceted role of quercetin in reversing cancer MDR, studies that support clinical uses of flavonoid are rare, most likely

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because of its pharmacokinetic properties including low stability, low solubility, poor cellular

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uptake and fast metabolism.13-14

Previously, we have shown that bioavailability of therapeutics can be significantly improved

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via conjugation with a stable delivery system for the selective targeting and elimination of cancer cells. We have also confirmed that multifunctional nanoparticle-based therapeutic

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systems can overcome drug resistance by neutralizing various resistance mechanisms whilst

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simultaneously enhancing the therapeutic outcome of anticancer agents.15-16 On the basis of

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these observations, QUR and DOX conjugated multifunctional nanoparticles were prepared herein for synergistic delivery of QUR and DOX to drug-resistant cancer cells. The new formulation of QUR could increase its potential and serve as a possible solution for its low bioactivity, thereby reducing chemoresistance while increasing the therapeutic efficacy of DOX for cancer therapy.

Considerable efforts have been devoted to improve novel multifunctional platforms toward the development of next-generation nanoparticles against drug resistance. They have been developed as an all-in-one biomedical platform with proposed configurations encompassing (1) cancer chemotherapeutics, (2) chemosensitizers (3) molecular (diagnostic) imaging agents and (4) active targeting components.17 Beyond these features, another important concern is the sensitivity of drug release in response to environmental stimuli at specific target sites. 4 Page 4 of 36

Therefore, the concept of stimuli-responsive nanoparticles has received much attention. Such nanoparticles are able to control drug-release after internalization, thus preventing drug leakage before reaching the tumor tissue, and improving the accumulation of

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chemotherapeutic agents to therapeutic levels within the target site.18 Considering the potential importance of these multiple functions within a single platform to

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overcome cancer MDR, I have rationally designed the bottom-up synthesis of pH-responsive

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Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles for combination therapy comprising four major components: (1) a luminomagnetic nanocomposite as contrast agent with both magnetic

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(Fe3O4) and optical contrast (FITC) serving as a dual imaging probe both in-vivo and in-vitro, (2) biotin (BTN) as a tumor specific targeting moiety to facilitate the delivery of nanoparticles

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to the tumor cells, (3) doxorubicin (DOX) as a model chemotherapeutic drug, and (4) quercetin (QUR) as a natural chemosensitizer aiming to modulate resistance-mediating

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factors. Fe3O4@SiO2(FITC)-BTN/QUR nanoparticles containing only chemosensitizer and

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Fe3O4@SiO2(FITC)-BTN/DOX nanoparticles containing only chemotherapeutic drug were

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also synthesized to evaluate the synergistic potential of different therapeutic combinations. The feasibility of nanoparticles was evaluated by investigating their capability in cellular uptake and intracellular persistence. Cytotoxicity was analyzed by comparing drug-sensitive A549 cells with drug-resistant A549/DOX cells, versus human pulmonary epithelial BEAS2B cells. I also examined these multifunctional platforms by flow cytometric analyses of apoptosis with 7-ADD/PE-annexin-V double staining and cell cycle with propidium iodide DNA staining to evaluate their MDR-modulatory effect on the sensitivities of A549 and A549/DOX cells to DOX chemotherapy.

Material and Methods

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Materials Iron (II) chloride tetrahydrate (FeCl2.4H2O) (99%), iron (III) chloride hexahydrate (FeCl3.6H2O) (98%), tetraethyl orthosilicate 99.9% (TEOS), fluorescein isothiocyanate

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(FITC), biotin (BTN), quercetin ≥98% (QUR), 3-aminopropyltriethoxysilane (APTES), N,Ndicyclohexyl-carbodiimide (DCC), N-hydroxysuccinimide (NHS), Glutaraldehyde 25%

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aqueous solution, FT-IR grade potassium bromide ≥99% (KBr), dimethyl sulfoxide (DMSO),

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triton X-100, 3-[4,5-dimethyl-2-thialzolyl]-2,5-diphenyltetrazolium bromide (MTT), trypsin, RNase A and propidium iodide (PI) were purchased from Sigma-Aldrich Chemicals. Oleic

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acid (99%), ammonium hydroxide 25% aqueous solution, 1-hexanol (>98%), cyclohexane and toluene were purchased from Fluka/Riedel-de Haën Chemicals. Doxorubicin was

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obtained from SABA Pharma. DMEM growth medium, 10% Fetal bovine serum (FBS), streptomycin, penicillin and L-glutamic acid were purchased from Gibco Life Technologies.

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7-aminoactinomycin (7-ADD) and PE-annexin-V were purchased from BD Biosciences. All

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in deionized Milli-Q water.

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other chemicals and reagents were of the highest purity. All the experiments were performed

Cell Cultures

A549 (human epithelial lung carcinoma) and BEAS-2B (immortalized human lung epithelial) cell lines were kindly provided by the Biotechnology and Bioengineering Research and Application Centre, Izmir Institute of Technology, Turkey. A549 and BEAS-2B cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, 100 U/mL penicillin and 2 mM L-glutamic acid. DOX-resistant A549 cells were isolated by stepwise selection of resistant cells upon culture with increasing concentrations of DOX (0.1/1/5/10/50/100 nM). Briefly, A549 cells 6 Page 6 of 36

were treated with DOX at increasing rates and when the cells were capable of growing and reaching appropriate confluency at a certain concentration, the medium was changed with the following DOX concentration for next selection of resistant cells. Finally, the A549 cells

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acquired DOX resistance and were cultured in medium containing 100 nM DOX and thereafter, named as A549/DOX. The final tolerated concentration of 100 nM DOX represents

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the mean plasma concentration of doxorubicin in patients under chemotherapy.19 To eliminate the effects related to long-term culture of cancer cells, A549 and BEAS-2B cells were

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cultured and maintained under the same conditions as the DOX-resistant A549 cells in the

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absence of doxorubicin. All cell lines were incubated in 5% CO2 and 90–100% relative

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humidity at 37 °C.

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Synthesis of Multifunctional Nanoparticles

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The parental Fe3O4@SiO2(FITC) nanoparticles were synthesized as described in our previous work.15 They consist of superparamagnetic iron oxide nanoparticles (as magnetic contrast

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agent), coated with layers of silica shells, encasing FITC within (as optical contrast agent), for imaging, biocompatibility and molecular functionalization. To ensure preferential tumor cell uptake of the nanoparticles, the outermost layer of Fe3O4@SiO2(FITC) was then functionalized with BTN molecules by silanization with BTN-APTES conjugate. Since biotin-specific uptake systems are highly over-expressed on the cell surface of rapidly proliferating cancer cells, biotin derivatization allows the selectively delivery of the nanoparticles to tumor cells in the presence of normal cells via receptor-mediated endocytosis.20 In brief, an APTES ester of BTN (BTN-APTES) was prepared by mixing biotin (8.0 mg) with APTES (2.0 µL) in 40 mL dry dimethyl sulfoxide (DMSO) in the presence of NHS (1.1 mg) and DCC (4.7 mg) as the catalyst, at room temperature for 2 h.

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After this, a mixture of Fe3O4@SiO2(FITC) nanoparticles (100 mg), BTN-APTES conjugate and free APTES (17 µL) in toluene (160 mL) were stirred at room temperature for 24 h to introduce BTN-APTES conjugate and free APTES on the surface of silica-coated

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nanoparticles by hydrolysis and condensation of APTES through silanization. Final products were collected by a magnet, washed with toluene and ethanol several times to remove any

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unreacted reactants, and dried in a vacuum oven at room temperature, overnight. In this step, besides vectorization of nanoparticles, the surfaces were simultaneously modified with free

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APTES to form an amine-terminated overlayer for further functionalization.

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Fe3O4@SiO2(FITC)-BTN/QUR/DOX were prepared by conjugating QUR and DOX complexes on the surface of Fe3O4@SiO2(FITC)-BTN/NH2 nanoparticles via glutaraldehyde

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activation. For pH-responsive drug release, QUR and DOX complexes were covalently linked to amine-functionalized silica surface of nanoparticles via pH-labile acetal21 and imine22

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formation from the two hydroxyl groups on the catechol moiety of QUR and the amino sugar

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moiety of DOX, respectively. These acid sensitive linkages are stable at natural pH (∼ 7.4),

but broken at mildly acidic pH (∼ 5.0), which allow for the release of QUR and DOX in the

more acidic endosome environment (pH 5.0) versus systemic circulation pH (7.4). Thus endow the Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles with pH-switched QUR and

8 Page 8 of 36

DOX

release

property.

In

this

step,

both

Fe3O4@SiO2(FITC)-BTN/QUR

and

Fe3O4@SiO2(FITC)-BTN/DOX nanoparticles were also synthesized to evaluate synergistic potential of therapeutics. Briefly, the surface of Fe3O4@SiO2(FITC)-BTN/NH2 nanoparticles

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(10 mg) was activated in 20 mL 1.0% glutaraldehyde solution under vigorous mechanical stirring at room temperature for 1 h. Nanoparticles were subsequently collected via

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centrifugation, and the unreacted glutaraldehyde removed by extensive washing with ultrapure water. Glutaraldehyde activated nanoparticles were incubated with QUR (100 µM)

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and/or DOX (10 µM) complexes (1.0% DMSO) in 20 mL PBS solution (pH 7.4) under

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vigorous mechanical stirring at room temperature for 6 h. An excess amount of QUR with low-dose DOX concentration (molar ratio of QUR to DOX = 10) was used to evaluate the

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sensitization effect of QUR and to reveal low-dose DOX effects against resistant cancer cells. The amount of bound QUR or DOX was calculated from the difference between the amount

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of QUR or DOX introduced into the coupling reaction mixture, and the amount of QUR or

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DOX present in the washing water after immobilization. This was carried out by measuring QUR and DOX absorbance at 380 nm and 480 nm. The resulting nanoparticles: (1)

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Fe3O4@SiO2(FITC)-BTN/QUR;

(2)

Fe3O4@SiO2(FITC)-BTN/DOX

and

(3)

Fe3O4@SiO2(FITC)-BTN/QUR/DOX were magnetically separated and washed with 1% DMSO in PBS several times to remove any unreacted reactants and dried under vacuum at room temperature, overnight.

Structural and Physicochemical Characterization Dynamic light scattering (DLS) measurements were performed at 25°C, using a Malvern Zetasizer NanoZS compact scattering spectrometer. Average hydrodynamic diameters, size distributions and surface charge analysis of the samples were determined using Malvern

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Dispersion Technology Software 7.11. Nanoparticles were suspended in ultrapure water to give optimum signal intensity. All measurements were repeated five times to verify the reproducibility of the results.

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The Fourier transform infrared spectroscopy (FTIR) spectra of the nanoparticles were collected with a “PerkinElmer Spectrum-100” spectrophotometer in the range 450–4000 cm-1.

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The spectra of the dried samples were obtained by employing a KBr pellet.

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Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) studies were performed with a high resolution environmental scanning electron microscope

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(FEI Quanta 250 FEG) equipped with a Schottky field emission gun (FEG) for optimal spatial resolution and an energy-dispersive X-ray spectrometer (Oxford AZtec). Prior to examination,

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the lyophilized nanoparticles were placed on a double stick tape over aluminum stubs to get a

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uniform layer of particles.

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Scanning transmission electron microscopy (STEM) images of the nanoparticles were obtained with a “FEI Quanta 250 FEG” microscope operating with STEM Detector. The

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nanoparticles were dispersed in water under sonication and a drop was placed on a carboncoated 400 mesh copper grid followed by air-drying.

In vitro Release Studies

The release of drugs from the nanoparticle formulations was examined by the dialysis bag diffusion

method.

The

aqueous

suspensions

of

Fe3O4@SiO2(FITC)-BTN/QUR,

Fe3O4@SiO2(FITC)-BTN/DOX or Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles (1 mg/mL) were placed into the respective dialysis bags (MWCO 3500 Da) and suspended in 25 mL of 1% DMSO in PBS buffer solution with pH 5.0 or 7.4 at 37°C under gentle magnetic

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stirring, respectively. Then, 1 mL of the each release medium was withdrawn at predetermined time points and replaced with 1 mL of fresh buffer solution of relevant pH. The amount of QUR and DOX in the withdrawn samples was determined using UV-vis

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spectrometry, at the corresponding wavelengths. All experimental procedures were repeated three times to obtain the average value. The cumulative release of QUR or DOX was

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calculated according to the following equation:

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The cumulative release of QUR or DOX (%) = (mass of released QUR or DOX / total mass of

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conjugated QUR or DOX) x 100

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Cellular Uptake Analysis

The cellular uptake behavior of the nanoparticle formulations was investigated in drug-

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sensitive A549, drug-resistant A549/DOX and non-tumoral BEAS-2B cells using

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fluorescence microscopy and flow cytometry. For microscopic observation, all cells (1 × 105

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cells/well) were seeded in 12-well plates overnight to allow the adhesion of the cells before experiments. Next, the nanoparticle formulations were added to the incubation medium at a concentration of 10 µg/mL for 4 and 24 h of incubation to investigate time-dependent uptake and MDR-modulatory effect in 5% CO2 at 37°C. After incubation, all cells were washed three times with PBS and later microscopic images in the green channel, for detection of the FITC label encapsulated in nanoparticles, and in the bright-field were obtained by fluorescence microscopy. Internalization of the nanoparticles was visualized using an Olympus IX2ILL100 fluorescence microscope equipped with an appropriate filter set. Images were acquired using a CCD camera and analyzed using ImageJ advanced version software. For cellular uptake quantitative analysis, the cells (2 × 105 cells/well) were seeded in 6-well plates overnight before experiments. The medium from each well was discarded and cells were 11 Page 11 of 36

treated as described above. The samples were trypsinized and harvested to obtain a cell suspension, which was then analyzed for the distribution of FITC fluorescence by flow

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cytometry.

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Cytotoxicity of Multifunctional Nanoparticles

The cytotoxicity of the nanoparticle formulations was evaluated by MTT assay. A549,

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A549/DOX and BEAS-2B cells were seeded into 96-well plates at a density of 1x104 per well

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and grown overnight. The cells were then incubated with increasing concentrations (1.0/10/50/100/200 µg/mL) of the nanoparticle formulations, free DOX (100 nM), free QUR

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(10/100 µM), and the combinations of free DOX (100 nM) and QUR (10/100 µM), respectively, in 100 µL of growth medium with 1% DMSO for 48 h at 37°C under 5% CO2.

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Following this incubation, cells were incubated in medium containing 0.5 mg/mL of MTT for

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4 h. The medium was discarded, and the precipitated formazan violet crystals were dissolved in 150 µL DMSO to solubilize the formazan. After shaking the plate for 10 min, the

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absorbance of the sample was measured at 570 nm by multidetection microplate reader. The absorbance of dissolved formazan in the visible region correlates with the number of viable cells. The percentage of viable cells was calculated according to the following equation: Cell viability (%) = (Mnanoparticles/free drugs – Mblank) / (Mcontrol – Mblank) x 100 where Mnanoparticles/free drugs is the absorbance of the cells, growth medium and nanoparticles or free drugs, Mcontrol is the absorbance of the cell and growth medium, and Mblank is the absorbance of the growth medium alone. Cytotoxicity was evaluated with reference to the IC50 value that was defined as the concentration of compound causing death in 50% of cells. IC50 values were calculated from dose-response curves (nanoparticle concentration vs. cell survival fraction) obtained in repeat experiments. 12 Page 12 of 36

Detection of Apoptotic Cells by Flow Cytometry

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The percentage of cells undergoing apoptosis induced by the nanoparticle formulations was measured using flow cytometry with 7-ADD and PE-annexin-V double staining. A549 and

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A549/DOX cells (1×105 cells/well) were seeded in 6-well plates with 2 mL of growth medium overnight before experiments. Fe3O4@SiO2(FITC)-BTN/QUR, Fe3O4@SiO2(FITC)-

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BTN/DOX or Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles were added to the incubation medium at IC50 concentrations and then the cells were incubated for 4 h in 5% CO2

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at 37 °C to allow uptake of the nanoparticles. Before analysis, the cells were carefully washed with cold PBS, digested with trypsin and collected by centrifugation. The cells were washed

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twice with cold PBS, resuspended in 200 µL of annexin binding buffer and stained with 10 µL of 7-ADD and PE-annexin-V. The stained cells were first incubated for 15 min at room

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temperature in the dark, and then analyzed by flow cytometry. The untreated cells incubated

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with medium alone were used as the controls. Unstained cells, cells stained with PE-annexin-

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V alone and cells stained with 7-AAD alone were used to set up compensation and quadrants. Flow cytometric analysis was performed on a FACS (Facscanto; Becton Dickinson, San Jose, CA) by counting 10,000 events.

Cell Cycle Analysis by Flow Cytometry To

determine

the

antiproliferative

Fe3O4@SiO2(FITC)-BTN/DOX

or

effects

of

Fe3O4@SiO2(FITC)-BTN/QUR,

Fe3O4@SiO2(FITC)-BTN/QUR/DOX

nanoparticles

against A549 and A549/DOX cells, cell cycle distributions were analyzed by flow cytometrybased propidium iodide (PI) staining. The cells (1×105 cells/well) were seeded in 6-well plates with 2 mL of growth medium overnight before experiments and then incubated with IC50 13 Page 13 of 36

concentrations of the nanoparticle formulations for 24 h at 37 °C under 5% CO2. After incubation, the cells were trypsinized, washed once with PBS and fixed in 80% ethanol overnight at -20°C. The next day, after centrifugation of fixed cells, the pellet was washed

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with cold PBS and then resuspended in DNA staining solution [200 µL 0.1% Triton X-100 in PBS, 20 µL (200µg/mL) RNase A and 20 µL (1mg/mL) propidium iodide] for 30 min at

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room temperature. The stained cells were analyzed using flow cytometer. The fraction of the

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G0-G1, S and G2-M phase of cells on DNA histograms was analyzed by ModFit software.

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Statistical Analysis

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All data were represented as means ± standard deviation (SD). Statistical analysis was performed with the Student's t test, using Excel Software (Microsoft). A P value of ≤0.05 was

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Results and Discussion

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considered statistically significant.

Synthesis and Characterization of Multifunctional Nanoparticles The

Fe3O4@SiO2(FITC)-BTN/QUR/DOX

nanoparticles

consisted

of

silica-coated

luminomagnetic nanomaterials, triple conjugated to BTN, QUR and DOX. To construct the Fe3O4@SiO2(FITC)-BTN/NH2 nanoparticles, superparamagnetic (Fe3O4) nanocrystals were synthesized by the co-precipitation method and coated with two silica shells for the incorporation of FITC by microemulsion sol–gel chemistry. The cancer targeting feature was then endowed by covalent bonding BTN on the surface of nanoparticles by means of an esterification reaction. Lastly, the silica surface was conjugated with QUR and DOX by pHlabile acetal and imine linkages for environmentally responsive drug release, with the 14 Page 14 of 36

expectation that these acid-sensitive linkages would promote QUR and DOX release into the more acidic endosome of cancer cells instead of in the systemic circulation environment. The quantitative study of QUR and DOX conjugation on the silica surface showed that 6.12 µΜ of

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QUR and 0.59 µM of DOX were co-conjugated per mg Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles, while coupling yields of 7.67 µΜ of QUR per mg Fe3O4@SiO2(FITC)-

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BTN/QUR and 0.94 µM of DOX per mg Fe3O4@SiO2(FITC)-BTN/DOX were obtained (Table 1). The designed synthesis and anti-MDR strategy of the nanoparticles are

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schematically illustrated in Figure 1.

Figure 1. Schematic representation of Fe3O4@SiO2(FITC)-BTN/QUR/DOX synthesis, targeted cell uptake and intracellular drug release. 15 Page 15 of 36

The mean hydrodynamic size of the nanoparticle formulations were 32.4 ± 5.2, 47.9 ± 6.7, ± 7 and 59.5 ± 8.4 nm for Fe3O4@SiO2(FITC)-BTN/NH2, Fe3O4@SiO2(FITC)-

BTN/QUR,

Fe3O4@SiO2(FITC)-BTN/DOX

and

Fe3O4@SiO2(FITC)-BTN/QUR/DOX

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55.2

nanoparticles, respectively, with narrow size distribution as demonstrated in Fig. 2a. To

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improve the biodistribution and circulation time of nanoparticles in the bloodstream, the sizes

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of the nanoparticles are of critical importance. Particles with a diameter ranging from 10-100 nm are optimal to prevent the uptake of nanoparticles by the ReticuloEndothelial System

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(RES) as well as for the endocytosis behavior of tumor cells.23 The results suggest that these particles are small enough to carry and deliver their therapeutic payloads to cancer cells.

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Table 1. Immobilization yields, size and zeta potential of the nanoparticle formulations. QUR and DOX

Formulations

Immobilization

Immobilization

Concentration

Yield (%)

(µM per mg

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QUR and DOX

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Nanoparticle

Size (nm)

Zeta Potential (mV)

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nanoparticles)

Fe3O4@SiO2(FITC)

-

-

32.4 ± 5.2

+8.6 ± 3.3

76.7

7.67

47.9 ± 6.7

-14.2 ± 2.2

94.3

0.94

55.2 ± 7.0

-14.7 ± 2.0

Fe3O4@SiO2(FITC)

61.2 (QUR) /

6.12 (QUR) /

-BTN/QUR/DOX

59.5 ± 8.4

-17.2 ± 2.8

58.7 (DOX)

0.59 (DOX)

-BTN/NH2

Fe3O4@SiO2(FITC) -BTN/QUR

Fe3O4@SiO2(FITC) -BTN/DOX

The zeta potential of nanoparticles was analyzed to confirm the change in their surface potential due to proper bioconjugation at different stages of synthesis (Figure 2a and Table 1).

16 Page 16 of 36

The Fe3O4@SiO2(FITC)-BTN/NH2 nanoparticles exhibited a positive zeta potential (ζ) value of +8.6 (pH 7.4) due to presence of excess positively charged amino groups, which nullify the BTN charge and make the overall surface charge slightly positive.15 Further surface

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functionalization of the nanoparticles by conjugating QUR and/or DOX led to an obvious charge reversion to -14.2, -14.7 and -17.2 mV values (pH=7.4) for Fe3O4@SiO2(FITC)Fe3O4@SiO2(FITC)-BTN/DOX

and

Fe3O4@SiO2(FITC)-BTN/QUR/DOX

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BTN/QUR,

nanoparticles, respectively, attributable to a function of the high ionizability of QUR and

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DOX groups on silica surface. The negatively charged properties of the nanoparticle

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formulations could reduce RES-mediated clearance and eliminate the adsorption of plasma protein; thereby, enhancing the blood circulation time with improved pharmacokinetic of

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therapeutics, which are essential for their biological applications.

In order to evaluate the success of the surface functionalization with BTN, QUR and DOX,

FTIR

spectra

of

Fe3O4@SiO2(FITC)-BTN/NH2,

Fe3O4@SiO2(FITC)-BTN/QUR,

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the

d

Fourier transform infrared (FTIR) spectroscopy was used. For comparison, Figure 2b gives

Fe3O4@SiO2(FITC)-BTN/DOX and Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles. The

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appearance of the absorption peaks at 1214 and 1525 cm-1 in the Fe3O4@SiO2(FITC)BTN/NH2 FTIR spectrum confirms the existence of biotin24 and the primary amine group of APTES15 moieties, respectively. On the other hand, FTIR analysis of Fe3O4@SiO2(FITC)BTN/QUR,

Fe3O4@SiO2(FITC)-BTN/DOX

and

Fe3O4@SiO2(FITC)-BTN/QUR/DOX

nanoparticles shows the presence of new bands at 1385 and 1570 cm−1, which are attributed to bending of the O–H bond in aromatic hydrocarbon phenolic groups of QUR25 and the stretching vibration of two carbonyl groups of the anthracene ring of DOX15, respectively, indicated that QUR and DOX were successfully attached to the surface of the Fe3O4@SiO2(FITC)-BTN/NH2 nanoparticles.

17 Page 17 of 36

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Figure 2. (A) Size distributions and zeta potential measurements of the nanoparticle

Ac ce p

formulations obtained at pH=7.4 using DLS. (B) FTIR characterizations of the nanoparticle formulations. (C) EDX spectra of the nanoparticles (inset: weight percentages of composition), obtained during SEM experiments. The untagged Al peak is from the double stick aluminum tape used in sample preparation.

The exterior morphology and interior structure of the synthesized Fe3O4@SiO2(FITC)BTN/QUR/DOX nanoparticles were examined by SEM and STEM; and the respective chemical compositions at different fabrication stage were determined by EDX. SEM and STEM images show that the nanoparticles have nearly spherical shape and present a narrow size distribution (Figure 3a). The core-shell structures can be easily recognized due to good 18 Page 18 of 36

contrast between the cores and the shell regions. The white cores are magnetic particles and the gray part is the silica shell. The thickness of the shell of the nanoparticles in dry state was about 50 nm which is almost in accordance to those found by DLS, indicating that the coated

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nanoparticles are stable and well dispersed in solution without aggregation or accumulation. The EDX analysis of the nanoparticle formulations clearly showed the progressive increase in

cr

C content and reduction in N content on the silica surfaces due to organic functionalities

us

which are gradually enriched by iterative chemical conjugation (Figure 2c and Table 2).

Atomic (%)

The Nanoparticle Formulations

O 63.9

Fe3O4@SiO2(FITC)-BTN/QUR

54.2

d 56.3

te

Fe3O4@SiO2(FITC)-BTN/DOX

Si

C

N

Fe*

14.3

14.1

7.5

0.2

13.7

26.9

4.8

0.4

12.5

26.4

4.4

0.8

11.7

33.5

4.2

0.5

M

Fe3O4@SiO2(FITC)-BTN/NH2

Fe3O4@SiO2(FITC)-BTN/QUR/DOX *

an

Table 2. Surface atomic percentages of the nanoparticles, obtained from EDX spectra.

50.1

Ac ce p

Fe content is detected due to the penetration depth of the X-ray beam through the thick silica shell.

In vitro pH-stimuli Release Study The nanoparticles were formulated for pH-sensitive drug release to control the release of the conjugated therapeutics based on local pH levels. Thus, the nanoparticles are expected to retain the drug combination during circulation while actively releasing them inside the target tumor cells. For this reason, the ability of the nanoparticles to release the conjugated therapeutics at pH 7.4 and 5.0 was tested to simulate the physiological pH in the blood circulation and the acidic pH in endosome of tumor cells. Most of the drugs were released

19 Page 19 of 36

from the nanoparticle formulations within a few hours at pH 5.0, whilst the nanoparticles showed no more than 18% of the conjugated drugs release at pH 7.4 for 24 h, implying that pH-labile conjugation exerted a dominant influence on preventing the premature drug release.

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During the first 4 h under pH 5.0, the release of DOX from Fe3O4@SiO2(FITC)-BTN/DOX and Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles showed release percentages of 69%

cr

and 65% , respectively. After 24 h, the cumulative amount of DOX released increased to 75% and 73% from the corresponding nanoparticles. Furthermore, the release of QUR from and

Fe3O4@SiO2(FITC)-BTN/QUR/DOX

us

Fe3O4@SiO2(FITC)-BTN/QUR

nanoparticles

an

exhibited a similar release pattern to that of DOX: approximately 47% and 40% of QUR were released in 4 h under pH 5.0, whereas the cumulative percentages were improved to 64% and

M

61% after 24 h, respectively from the corresponding nanoparticles (Figure 3b). In general, the controlled transport of drugs has been mostly achieved by loading them into

d

nanocarriers. Even if these carriers entrap high amounts of therapeutics and accumulate inside

te

the tumor, they may not be able to release the drug to kill malignant cells (especially drug-

Ac ce p

resistant cells) in time, as the drugs slowly diffuse from the nanoparticle core.26 To eliminate this obstacle, conjugating the drugs to the nanoparticle surface with sensitive linkage could lead to fast intracellular drug release and a high level intracellular drug concentration to achieve the effective therapeutic concentration for reversing the MDR efflux function.

20 Page 20 of 36

ip t cr us an M d te

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Figure 3. (A) SEM and STEM (inset) images of Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles. (B) Cumulative percentages of released DOX and QUR under pH 7.4 and pH 5.0 over time from Fe3O4@SiO2(FITC)-BTN/QUR; Fe3O4@SiO2(FITC)-BTN/DOX; and Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles. (C) Cell viability of A549; A549/DOX; and BEAS-2B cells after 48 h of incubation with free DOX (100 nM), free QUR (10/100 µM), combination of free DOX (100 nM) and free QUR (10/100 µM), and the nanoparticle formulations. The nanoparticle concentrations are expressed as µg nanoparticles per mL. The results are expressed as percentage of cell viability or cell number obtained in the untreated controls. Each column represents the mean ± SD of three independent experiments performed in triplicate normalized to non-treated cells (taken as 100%).

21 Page 21 of 36

Cellular uptake

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The cellular uptake behaviors and resistance reversing abilities of nanoparticle formulations were examined in drug-sensitive A549, drug-resistant A549/DOX and non-tumoral BEAS-2B

cr

cells by fluorescence microscope and flow cytometry (Figure 4).

fluorescence

was

observed

from

the

images

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For drug-sensitive A549 cells with a relatively low expression of MDR proteins, green of

Fe3O4@SiO2(FITC)-BTN/QUR,

an

Fe3O4@SiO2(FITC)-BTN/DOX and Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles at 4 h with no significant differences with 74.2%, 70.3% and 73.7% cellular uptake efficiencies

M

respectively, which indicated that the vectorized nanoparticles with BTN successfully penetrated into the cells. After prolonging incubation periods to 24 h, the fluorescence

d

intensity (25.1%) from Fe3O4@SiO2(FITC)-BTN/DOX nanoparticles obviously decreased as

te

compared with both Fe3O4@SiO2(FITC)-BTN/QUR (63.4%) and Fe3O4@SiO2(FITC)BTN/QUR/DOX (61.8%) intensities. This reduction was due to overexpression of MDR

Ac ce p

proteins, which demonstrated that the sensitization effect of QUR was successfully overcoming drug resistance. On the other hand, in spite of the development of drug resistance in cancer cells, the weak green fluorescence, which was still visible in the cells incubated with Fe3O4@SiO2(FITC)-BTN/DOX nanoparticles after 24 h, corroborated that rationally designed nanoparticles are capable of minimizing drug expulsion by efflux pumps and leading to a persistent intracellular accumulation and effectiveness. For the drug-resistance A549/DOX cells, the fluorescence intensity of Fe3O4@SiO2(FITC)BTN/DOX nanoparticles (21.9% for 4 h; 14.5% for 24 h) was very low from Fe3O4@SiO2(FITC)-BTN/QUR (57.6% for 4 h; 45.4% for 24 h) and Fe3O4@SiO2(FITC)BTN/QUR/DOX (55.4% for 4 h; 47.1% for 24 h) nanoparticles in both 4 and 24 h incubation 22 Page 22 of 36

periods due to the high MDR efflux function. In contrast, the nanoparticle formulations conjugated with QUR showed consistent fluorescence which supports intracellular stability and accumulation of the nanoparticles even after 24 h, confirming QUR’s functional in

reversing

MDR.

Furthermore,

Fe3O4@SiO2(FITC)-BTN/QUR/DOX

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involvement

nanoparticle uptake was minimal in normal BEAS-2B lung cells during the 4 and 24 h

cr

incubations (9.3% and 18.8% respectively), confirming that the nanoparticles facilitate internalization into tumor cells but not normal cells. Overall, these results confirmed the

us

necessity of BTN and QUR in Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles to achieve

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true molecular targeting of cancer cells and inhibition of the MDR-efflux activity,

Ac ce p

te

d

M

respectively.

23 Page 23 of 36

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Figure 4. Cellular uptake quantitative analyses and fluorescence microscopy images of A549; A549/DOX; and BEAS-2B cells after incubation with (A) Fe3O4@SiO2(FITC)-BTN/QUR; (B)

Fe3O4@SiO2(FITC)-BTN/DOX

and

(C)

Fe3O4@SiO2(FITC)-BTN/QUR/DOX

nanoparticles for 4 and 24 h. For columns: 1, bright-field images; 2, fluorescence images; and 3, the merger of both. Scale bar: 40 µm.

Cytotoxicity of the Multifunctional Nanoparticles

24 Page 24 of 36

To examine the restoring effect of QUR on DOX cytotoxicity toward drug-sensitive A549, drug-resistant A549/DOX and non-tumoral BEAS-2B cells, the cell inhibitory effects of the nanoparticle formulations and free drug combinations were investigated using MTT cell

ip t

proliferation assay. Among all nanoparticles tested, Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles exhibited stronger cell inhibitory effects with approximately 3.8- and 29.2-fold

cr

lower IC50 values in A549 cells as compared to Fe3O4@SiO2(FITC)-BTN/DOX and Fe3O4@SiO2(FITC)-BTN/QUR nanoparticles, respectively. Importantly the nanoparticles

us

were notably more toxic (5.9-fold) than Fe3O4@SiO2(FITC)-BTN/DOX nanoparticles while

an

moderately more toxic (3.1-fold) than Fe3O4@SiO2(FITC)-BTN/QUR nanoparticles in A549/DOX cells, demonstrating the high potential of the nanoparticles for treating drug-

M

resistant lung cancer (Table 3). This data showed that Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles led to an obvious inhibition concentration reversion in drug-resistant

nanoparticles.

Moreover,

Fe3O4@SiO2(FITC)-BTN/QUR

nanoparticles

te

BTN/DOX

d

A549/DOX cells to the IC50 value used in drug-sensitive A549 cells for Fe3O4@SiO2(FITC)-

demonstrated a higher cytotoxicity in A549/DOX cells with an approximately 2.2-fold better

Ac ce p

IC50 value than A549 cells, thus confirming the function of QUR in reversing cancer MDR. Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles did not affect normal lung cell growth even at high concentration (200 µg/mL) and exhibited decreased DOX-mediated cytotoxicity as compared to Fe3O4@SiO2(FITC)-BTN/DOX nanoparticles (Figure 3c). In further cytotoxicity studies, the cells were exposed to 100 nM free DOX, representing the mean plasma concentration of doxorubicin in patients under chemotherapy, which was approximately 3

times

higher than

the DOX concentration

in

50 µg/mL of

Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles; to 10 µM and 100 µM free QUR, which correspond to approximately 30- and 300-fold increased QUR concentrations compared to 50 µg/mL

of

Fe3O4@SiO2(FITC)-BTN/QUR/DOX

nanoparticles,

respectively;

or

to

25 Page 25 of 36

combinations of 100 nM free DOX with 10 µM and 100 µM free QUR respectively. As shown in figure 3C, Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles with notably lower drug concentrations exhibited remarkably higher cytotoxicity than that exhibited by the

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combinations of the free DOX and free QUR in both A549 and A549/DOX cells. Free QUR showed more dose dependent inhibition in A549/DOX cells than in A549 cells, but was not as

cr

cytotoxic as the nanoparticle formulations in reversing cancer MDR. Furthermore, free QUR did not decrease the cytotoxic effect of free DOX with both concentrations in BEAS-2B cells

us

when compared with the nanoparticle formulations, most likely due to its low solubility.

an

We have previously demonstrated that neutralizing the action of survivin (BIRC5) potentiates the efficacy of DOX against chemoresistance.15-16 Similarly in this work, QUR, as a potential

M

survivin suppressor within its MDR-modulating activity11-12, not only potentiated the efficacy of DOX in A549 and A549/DOX cells but also sensitized drug resistant A549/DOX cells to

d

DOX chemotherapy, while attenuating DOX-mediated cytotoxicity in non-tumoral BEAS-2B

te

cells. Staedleret et al. also reported that QUR enhances the therapeutic efficacy of doxorubicin in highly invasive breast cancer cells and simultaneously reduces doxorubicin-induced toxic

Ac ce p

side effects.27 In addition, Kaiserova et al. showed that QUR lowers DOX-induced toxicity by chelating iron, inducing antioxidant activity and inhibiting carbonyl reductase.28 These observations suggest that Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles may be recognized as a promising approach to improve bioactivity of QUR and the therapeutic index of DOX, which are capable of inducing cytotoxicity in drug-resistant cancer cells, while producing negligible effects on normal cells. Table 3. The IC50 values of the nanoparticles in A549, A549/DOX and BEAS-2B cells when incubated for 48 h. N.A. = Not Applicable (the data cannot be fitted as not enough inhibition was observed) in the cells. Values represent the mean ± SD of three independent experiments.

26 Page 26 of 36

A549/DOX

BEAS-2B

Fe3O4@SiO2(FITC)-BTN/QUR

421.2 ± 8.4

191.7 ± 5.4

N.A.

Fe3O4@SiO2(FITC)-BTN/DOX

55.0 ± 3.7

362.1 ± 8.8

≥ 500

Fe3O4@SiO2(FITC)-BTN/QUR/DOX

14.4 ± 2.3

61.1 ± 4.1

N.A.

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A549

Nanoparticle Formulations (µg/mL)

cr

Detection of Apoptotic Cells by Flow Cytometry

us

To investigate the apoptosis-inducing ability of the nanoparticle formulations in drugsensitive A549 and drug-resistant A549/DOX cells, and to examine whether QUR can

analyzed

by

flow

cytometry

an

enhance sensitivity to DOX treatment, the percentage of cells undergoing apoptosis was with

7-ADD

and

PE-annexin-V

double

staining.

M

Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles, versus the other nanoparticles, strongly increased the percent of late apoptotic cells (ADD+/PE-V+) in A549 and A549/DOX cells to

d

93.9 and 69.8%. When cells were exposed to Fe3O4@SiO2(FITC)-BTN/DOX nanoparticles,

te

the number of late apoptotic cells in drug-resistant A549/DOX cells was 10.8%, which was significantly lower than the apoptosis rate (76.5%) in drug-sensitive A549 cells due to the

Ac ce p

high MDR activity. In addition, the number of early apoptotic cells (ADD-/PE-V+) in A549/DOX cells (35.3%) was 4.3 times higher than in A549 cells (8.2%), suggesting that MDR proteins interfered with the efficacy of DOX-chemotherapy and led to prolonged initial phase of apoptosis. On the other hand, treatment with Fe3O4@SiO2(FITC)-BTN/QUR nanoparticles showed considerable apoptotic effect in both A549 and A549/DOX cells with total apoptotic percentages of 66.3 and 76.7%, respectively (Figure 5a). Overall these results demonstrated that Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles clearly potentiate the DOX-induced apoptosis in drug-resistant A549/DOX cells by modulating MDR proteins, while synergistically sensitizing A549 cells to DOX-chemotherapy, confirming the potentiation effect of QUR on drug-mediated cytotoxicity in cancer cells.

27 Page 27 of 36

Cell Cycle Analysis by Flow Cytometry

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To further determine the possible involvement of nanoparticle formulations in the regulation of the cell cycle, with the expectation that synergistic inhibition of the cells would promote

cr

the antiproliferative effect, the cell cycle phase distribution of drug-sensitive A549 and drugresistant A549/DOX cells was examined by flow cytometry. Exposure of A549 cells to

us

Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles significantly depressed cell proliferation in the G2/M checkpoint (56.6%), compared to the Fe3O4@SiO2(FITC)-BTN/QUR (24.9%)

an

and Fe3O4@SiO2(FITC)-BTN/DOX (41.7%) nanoparticles. These data indicated that the effects of QUR and DOX conjugated nanoparticles in the antiproliferative action are more

M

significant than those of the nanoparticles conjugated with QUR and DOX alone. Importantly, the pattern of cell cycle distribution of drug-resistant A549/DOX cells treated with

d

Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles showed a considerably higher percentage

te

of cells at G2/M phase (35.7%) in comparison to untreated control cells (12.4%). Similar

Ac ce p

results for G2/M arrest (23%) were observed following Fe3O4@SiO2(FITC)-BTN/QUR nanoparticle treatment, whereas Fe3O4@SiO2(FITC)-BTN/DOX nanoparticles hardly arrested A549/DOX cells (14.2%) in G2/M phase of the cell cycle. These results indicate that QUR induce antiproliferative response better than DOX treatment, and their combination has superior antiproliferative activity against drug-resistant A549/DOX cells (Figure 5b). These results demonstrated that, besides its proapoptotic effects, the Fe3O4@SiO2(FITC)BTN/QUR/DOX nanoparticles also show antiproliferative activity in both A549 and A49/DOX cells via G2/M-phase arrest.

28 Page 28 of 36

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Figure 5. (A) Apoptosis analysis of A549 and A549/DOX cells by flow cytometry. PEannexin-V / 7-ADD double staining of cancer cells treated with IC50 concentration of the nanoparticles. Viable cells labelled with PE-annexin-V(-)/7-ADD(-), early apoptotic cells labelled with PE-annexin-V(+)/7-ADD(-) and apoptotic cells labelled with PE-annexin-V(+)/ 7-ADD(+) in flow cytometric graphics. (B) Effect of the nanoparticles on the cell cycle

29 Page 29 of 36

distribution of A549 and A549/DOX cells determined by flow cytometry-based PI staining.

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Non-treated cells were used as control.

Conclusions

cr

In summary, Fe3O4@SiO2(FITC)-BTN/QUR/DOX multifunctional nanoparticles were

us

fabricated for environmentally responsive co-delivery of QUR and DOX as a way of reversing MDR. The structures of the nanoparticles were confirmed by different

an

characterization techniques: DLS, FTIR, EDX, SEM and STEM. The conjugation of QUR and DOX to the surface of nanoparticles via pH-labile linkages led to 6.7- and 4.8-fold

M

improved release at pH 5 versus pH 7.4 respectively, an important improvement in this drug delivery model. These findings also show that the conjugation of QUR on the surface of

d

nanoparticles helped to increase the bioavailability of QUR in tumor cells which resulted in

te

an increased chemosensitivity of drug-resistant cells to DOX chemotherapy and rendered

Ac ce p

improved pharmacokinetic and drug performance properties. Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles potentiated the efficacy of DOX with higher sensitivity in drug-resistant A549/DOX cells compared to its parent counterpart A549 cells, while attenuating DOX-mediated cytotoxicity in non-tumoral BEAS-2B cells. Cellular uptake studies confirmed that the designed nanoparticles are capable of minimizing drug expulsion by efflux pumps and lead to a persistent intracellular accumulation. Moreover, it was found that the combination effect of DOX and QUR within a multifunctional platform maximized apoptosis and G2/M-phase cell cycle arrest in both A549 and A549/DOX cells, compared to treatment with nanoparticles containing only QUR or DOX alone, thereby confirming the synergistic effect of this combination in reduction of resistance-mediating factors. In particular, the conjugation of QUR with DOX led to approximately 6- and 10-fold 30 Page 30 of 36

decreases in DOX concentrations against drug-sensitive A549 and drug-resistant A549/DOX cells, respectively. This reduction allowed the use of a low-dose chemotherapeutic agent without the need for dose increment; a significant finding in a clinical context.

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These observations show that the Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles orchestrate multiple functions simultaneously, including: a) enhancing DOX uptake and

cr

bioavailability of QUR into tumor cells via active targeting strategy and nanotechnology; b)

us

the pH-responsive nanoparticles can readily activated within tumor cells via its acid-sensitive drug release profile; c) reducing DOX efflux from tumor cells which is often mediated by

an

ABC transporter family; d) modulating signaling pathways to sensitize tumor cells to DOX chemotherapy and e) activating the antioxidant defense of normal cells against DOX

M

cytotoxicity via the inherent property of QUR. Overall, it can be concluded that this therapeutic strategy which combines QUR and DOX within a multifunctional platform might

Ac ce p

Acknowledgments

te

d

be promising against MDR by increasing the therapeutic index of chemotherapeutics.

I would like to thank Professor Anne Frary at the Izmir Institute of Technology for proofreading the manuscript and all members of The Center for Materials Research and Biotechnology and Bioengineering Center of Izmir Institute of Technology for their technical support.

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 31 Page 31 of 36

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(2004) On the formulation of pH-sensitive liposomes with long circulation times. Adv. Drug Delivery Rev. 56, 947−965.

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Research highlights

The pH-responsive multifunctional nanoplatform is proposed for reversal of cancer MDR.

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The low bioactivity of quercetin is eliminated with its new conjugated formulation.

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The pH-responsive drug release property improves the endosomal drug accumulation against MDR.

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d

M

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Graphical Abstract

36 Page 36 of 36